The goal of the proposed research is to develop HIV1 inhibitors based on the SAR-by NMR technology. The aims are to inhibit the interactions of CD4 with gp10 and gp120 with chemokine receptors, and to interfere with the function of gp41. The drug discovery process with include NMR-based screening of compound libraries that will be available through industrial and academic collaborations and subsequent development of the leads into HIV inhibitors. This component will focus on the NMR component while protein production and testing of biological activities will be carried out in the Core. The screening process will target the gp120 binding site of HCD4, the CD4 binding site of gp120, the chemokine receptor binding site of gp120, and gp41. This will require to have forms of these proteins available that are suitable for NMR spectroscopy. This is in place for hCD4/1-183. The NMR spectrum is already completely assigned and a high precision solution structure has been determined. NMR-based screening of libraries targeting CD4 is in progress. The wild-type of gp120 is not suited for NMR based drug discovery. Thus, we will use truncation and mutation strategies to produce glycan free fragments of gp120 that retain affinity to CD4 or chemokine receptors and are of a size amenable for NMR studies. A strategy will be adapted we have successfully applied to reduce the 50 kDa extracellular glycosylated domain of hCD58 to a glycan-free 10 kDa fragment which fully maintains computer-receptor binding function. The glycan-free gp120 mini-proteins will be used as targets for SAR-by-NMR investigations to identify molecules that will inhibit the interactions of gp120 with CD4 and chemokine receptors, respectively. A trimeric gp41 fragment will be another target for NMR-based identification of HIV inhibitors. We will start out with a protein construct that represent the fusion form of gp41. It shows good NMR spectra and is suitable for NMR analysis. Discovery and development of inhibitors will be targeted to a cavity of the inner trimeric core which accommodates side chains of the outer helical side chains of the outer helical layer in the fusion form of gp41.